January 1, 2014;
GRIN2B mutations in West syndrome and intellectual disability with focal epilepsy.
To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. We revealed de novo mutations in GRIN2B
encoding the NR2B
subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B
ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B
splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg(2+) block and higher Ca(2+) permeability, leading to a dramatically increased Ca(2+) influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. We identified GRIN2B
gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis.
MR/J004049/1 Medical Research Council
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Figure 1. Location of GRIN2B mutations in a schematic illustration of the conserved domains of the NR2B subunit (SP = signal peptide; ATD = amino-terminal domain, involved in receptor assembly; S1 and S2 form the ligand-binding domain; Pore = re-entrant pore-forming and transmembrane spanning domains; PDZ = PDZ domain binding motif). All
reported de novo mutations and their according phenotypes (ASD = autism spectrum disorders; FE = focal epilepsy; ID = intellectual disability; LGS = Lennox–Gastaut syndrome; Scz = schizophrenia) are listed in the top row. Mutations causing phenotypes without seizures are labeled in black, mutations in epilepsy patients are in red. So far, no
pathogenic variants have been observed in the C-terminal region of NR2B. Mutations causing West syndrome cluster within re-entrant pore-forming domain, whereas the mutation causing ID and focal epilepsy was observed in the glutamate-binding domain S1, similar to a recently described LGS case. Nonsynonymous variants that are believed not to be associated with abnormal phenotypes (gray) and are reported more than once (in brackets) in the Exome Variant Server (EVS) are listed in the bottom line.
Figure 2. Structural and functional consequences of missense mutations in GRIN2B. (A) Topology model of an NR1 and an NR2B subunit. Positions of the alterations p.Arg540His, p.Asn615Ile and p.Val618Gly are indicated by asterisks in the NR2 subunit consisting of an amino-terminal domain (ATD), the ligand-binding domain (LBD) including the S1 and S2 peptide segments, 3 transmembrane segments (M1, M2, and M3), a re-entrant pore loop (P), and an intracellular carboxy-terminal domain (CTD). Residue Arg540 lies within the glutamate-binding
domain, and Asn615 and Val618 in the ion channel pore. N = NH2-terminus; C= COOH-terminus. (B) Model of the transmembrane arrangement of the N-methyl-D-aspartate (NMDA) receptor composed of NR1 (green) and NR2B (cyan) subunits (top view). The arrow highlights the side chains of p.Asn615Ile and p.Val618Gly in the pore-forming region. (C) Gradual loss of Mg2+ inhibition of NR1-NR2B wild-type and
NR1-NR2B mutant receptor currents at −70mV. Respective sample traces of NR1-NR2B and NR1-NR2BAsn615Ile are shown above with inhibition of receptor currents by 1mM Mg2+ of NR1-NR2B (96 ± 0.9%, n=4) and mutant NR1-NR2BAsn615Ile (14 ± 7.2%, p < 0.0001, n
= 3), NR1-NR2BVal618Gly (48 ± 6.5%, p = 0.0003, n = 3), and NR1-NR2BArg540His (81 ± 3.2%, p = 0.005, n = 5) receptors. (D) Effect on Ca2+ permeability of NR1-NR2B wild-type and NR1-NR2B mutant receptor currents. Current–voltage relationships of NR1-NR2B receptors in the absence of Mg2+ in Na+-free extracellular solution reveal significant differences in the reversal potential (indicated by arrows) of NR1-NR2B (−31 ± 1.7mV, n = 4, black triangles) and mutant NR1-NR2BAsn615Ile (−1.0 ± 6.8mV,p = 0.004, n = 3, red squares),
NR1-NR2BVal618Gly (−5.4 ± 3.7mV, p < 0.001, n = 3, green squares), and NR1-NR2BArg540His (−9.4 ± 6.5mV, p = 0.013, n = 3, blue squares) receptor currents. (NMDG-Cl, N-methyl-D-glucamine chloride) Calculation of the relative divalent to monovalent cation permeability PCa/PNa by the Goldman–Hodgkin–Katz voltage equation revealed a >3-fold increase in Ca2+ permeability of the mutant NMDA receptors
(PCa/PNa for NR1-NR2B = 0.86; NR1-NR2BAsn615Ile = 5.22; NR2BVal618Gly = 3.12; and NR2BArg540His = 3.23).
Figure 3. Structural and functional analyses of the glutamate binding-domain mutation Arg540His. (A) Residue 540 is predicted to be located within the glutamate-binding S1 domain, and is significant in the stabilization of the tertiary structure of the glutamate-binding domain. (B) Substitution p.Arg540His is likely to abolish hydrogen bonding (blue
lines) with the backbone of Cys746 and His802 and a cation–pi interaction with His802, possibly leading to a relaxed fold in this region. (C) Pharmacological characterization of the apparent agonist affinities of wild-type NR1-NR2B (black triangles) and mutant NR1-NR2BArg540His
(red squares) N-methyl-D-aspartate receptors measured after heterologous expression in Xenopus laevis oocytes by 2-electrode voltage-clamping revealed that similar glutamate concentrations were required to elicit half-maximal responses (EC50 values = 0.72 ± 0.22μM and 0.31 ± 0.02μM, respectively, p = 0.14, n = 3). (D) Arg540 is a highly conserved residue within NR2A–D subunits.